Over the past decade, monoclonal antibodies achieved significant success and tremendous growth in bio-pharmaceutical industry, as well as the entire pharmaceutical industry. Compared with the traditional small molecule drugs, monoclonal antibodies possess many advantages including good specificity, significant therapeutic effect, less side effects, small administration amount, and so on. Regarding the characteristics of drug molecule, the antibodies have greater heterogeneity. This property of the antibodies is caused by a variety of factors, of which post-translational modification is the most important internal factor. Common post-translational modifications include antibody glycosylation, N terminal pyroglutamination, C terminal delysination, deamidizatioin, oxidation, isomerization and the like. Post-translational modifications will be detected and analyzed in many steps during antibody drug development, such as molecular identification, process development, quality control, etc.
IgG antibody glycosylation occurs at asparagine in heavy chain Fc region, which belongs to N-glycosylation, and an important structural components of the antibody. The core unit of IgG sugar chain is formed by connecting bifurcating structures formed by two N-acetylglucoses and three mannitoses. According to the differences in terminal galactose, core fucose, terminal sialic acid or the like, a variety of sugar chain structures can be constructed. IgG glycosylation is uneven because of different glycoform and content. The differences in glycosylation may affect the biological activity and pharmacokinetic characteristics of the antibody, such as CDC, ADCC, in vivo elimination half-life and the like.
When the N-terminal amino acid of the IgG antibody is glutamine, cyclization occurs readily to produce pyroglutamic acid, pyroE. The reaction can be spontaneous, or may be conducted under enzyme catalysis conditions. At C terminus of the IgG antibody molecule, de-lysine (—K) reaction occur readily. In most cases, both have no effect on biological activity of the antibody, but it is also reported that the N-terminal pyroglutamination of some antibodies may affect their antigen binding force. In addition, N terminal pyroglutamination and C terminal de-lysination will affect the charge distribution of the antibody, and charge characteristic is one of the important indicators of antibody quality control.
Therefore, it has important significance to establish an analysis method for rapid determination of IgG antibody glycosylation and terminal modification in the development of antibodies. Currently in the art, glycosylation and terminal modification are generally determined separately. Enzymatic quantitative fluorescence labeling method is a classic quantitative assay for determining IgG1 glycosylation, but the sample handling is quite complicated and time-consuming, and requires a large amount of sample. Mass spectrometry is also used for glycosylation analysis by detecting IgG enzymolysis fragments, such as papain enzyme and IdeS enzyme. However, these methods have some disadvantages, such as weak enzyme cleavage site selectivity, or high cost, or complicated sample handling, and hence they are inappropriate for batch testing of conventional or process development samples. Application of LC-MS in peptide mapping analysis is theoretically possible to determine the glycosylation and terminal modification of antibody at the same time, but there are many technical difficulties in the separation, determination and data analysis of sugar-containing polypeptides. Thus, it is inappropriate for quantitative analysis of glycosylation, and the sample handling is quite complicated and time-consuming, the digestion process may also have an impact on the existing terminal modification of antibodies. In immunoglobulin purification process, it is required to determine the protein purification status at any time, thus the large amount of sample required for detection and long detection time are still the biggest problems. Therefore, a suitable method for simultaneously and rapidly determining the glycosylation and terminal modifications of a small amount of IgG antibody during antibody development has not been reported yet.